1. Plasmids Chromosome Plasmid Plasmid + Transposon Plasmid + integron Plasmid+transposon +intergron Chromosome Chromosome + transposon Chromosome + transposon + integron

2. Antimicrobials 4: Testing and Selection Dr Fiona Walsh

3. Role of Antibiotic Therapy Prevention or Cure Cure or control Benefits outweigh disadvantages Efficient treatment –Test bacteria sensitivity –Understand antibiotic in human body

4. Objectives of lecture Sensitivity/Resistance testing methods Pharmacokinetics –Science of time course of drug in body –Increase effectiveness/reduce toxicity Pharmacodynamics –Relationship between drug concentration at site of infection and pharmacological response

5. Sensitivity tests Susceptible or resistant to antibiotic MIC = Minimum inhibitory concentration MBC = Minimum bactericidal concentration Minimum concentration required to inhibit growth –Disc diffusion –Agar dilution –Etest –Breakpoint MIC

6. Diffusion of antibiotic from a paper disc After Incubation Zone of Sensitivity Concentration of antibiotic at periphery of zone equals the MIC Disc Area of Bacterial growth Disc Concentration Gradient

7. 124816>16 Minimum Inhibitory Concentration (mg/L) 0 2 4 6 8 10 Number Breakpoint ( mg/L ) 0 4 1 8 2 16 Minimum Inhibitory Concentration (MIC) Determination

8. MIC Breakpoint: concentration above which the isolate is described at resistant and below which is susceptible e.g. S < 8mg/L R ≥ 8mg/L Breakpoint = 8mg/L Range: Lowest to highest MIC for population MIC 50 Median for series of MICs MIC 90 –MICs of population ordered from lowest to highest –MIC value of the strains that appears 90% up the series. –Antibiotic considered to be successful if > 90% of population inhibited. –Also show if resistance is emerging i.e. 10% of population resistant.

9. Minimum Inhibitory Concentrations MIC 50, MIC 90 and Range MIC 50 MIC 90

10. Etest Determination of MIC

11. Breakpoint Test to Determine Bacterial Sensitivity

12. Evaluation of Laboratory Tests MIC test on plates is the best –Time consuming and costly –Most detailed Disc test/Etest is easiest –Requires more skill to interpret Breakpoint –Least skill required –Technique must be exact –Can be read by computer –Large amounts of data

13. Minimum Bactericidal Concentration MIC 6432168421mg/l Subculture onto drug-free agar 168421mg/l MBC

14. Pharmacokinetics/Pharmacodynamics General terms for any drug, not antibiotic specific The term pharmacokinetics is used to define the time course of drug absorption, distribution, metabolism and excretion. The term pharmacodynamics refers to the relationship between drug concentration at the site of action and pharmacologic response. –However, when we apply these principles to antimicrobial therapy there are a number of factors that can alter the predicted outcome of therapy.

15. Factors which can influence therapeutic outcome BacterialHuman Inhibitory activity Absorption Subinhibitory activity Distribution Concentration-dependent activity Metabolism Time-dependent activity Excretion Bactericidal/bacteriostatic activity Protein-binding Post-antibiotic effect Resistance

16. Phamacokinetic Definitions Clearance is the removal of the drug from plasma and relates the rate at which a drug is given and eliminated to the resultant plasma levels (volume/time) C max is the maximum concentration reached at the site of infection, usually taken as the peak serum level. t max is the time taken, after dosage, to reach the C max. Half-life (t ½ ) is the time taken for the concentration of the drug in the plasma to decrease by half. This is often used as an indicator as to how often the drug should be administered.

17. Phamacokinetic Definitions Area Under the Curve (AUC) is the parameter that links clearance to dosing. It is easily calculated: Initial concentration / Elimination rate constant. Area Under the Inhibitory Curve (AUIC) is an antimicrobial adaptation of AUC, it refers to the concentration of the drug which is able to exert antibacterial activity over a given organism for a specific time. The AUIC is the drug concentration (AUC) divided by the MIC 90 for a specific bacterial species.

18. Pharmacokinetics Concentration (mg/L) Time (hours) t max t½t½ Dosing interval 64 8 16 32 4 2 1 C max

19. Pharmacokinetics Area Under the Curve Concentration (mg/L) Time (hours) 20 30 10 Area under the curve MIC 90 AUIC Preferably  250 but usable if > 125 AUC = Initial concentration / Elimination rate constant AUIC = AUC ( drug concentration) / MIC 90

20. Half-lives The half-life of the early antibiotics were quite short, perhaps only one hour or so. Therefore the antibiotic had to be administered many times per day. With oral versions, this causes problems with patient compliance and with parenteral versions, this becomes expensive in resources. Increasingly, the newer antibiotics have much longer half- lives, some up to 33 hours. This means that the patient needs to be dosed just once a day in order to maintain sufficient drug concentrations.

21. Toxicity The Need to Monitor Serum Levels Concentration (mg/L) 051015202530 Time (hours) Initial dose 64 8 16 32 4 2 1 Dosing interval

22. Antibiotic Assays Concentration (mg/L) Time (hours) 64 8 16 32 4 2 1 Initial dose Dosing interval Trough Peak

23. Post-Antibiotic Effect (PAE) Removal of Antibiotic Viable Count (cfu/ml) Control 1.6 hours to increase 1 log 10 1 log 10 increase 3.1 hours to increase 1 log 10 Antibiotic Induced death PAE = 3.1 - 1.6 = 1.5 hours Due to antibiotic effect only PAE = 3.1 - 1.6 = 1.5 hours Due to antibiotic effect only

24. Quantification of Post-Antibiotic Effect (PAE) The standard equation for PAE is: PAE (hours) = T - C T = is the time required for the count of cfu to increase 1 log 10 (10- fold) above the count immediately seen after drug treatment C = is the time required for the count to increase 1 log 10 in an untreated control culture PAE measures the time to reach normal logarithmic growth

25. Post-Antibiotic Effect Precise mechanism is still not understood Examples of PAE

26. Summary Sensitivity testing –Advantages –Disadvantages Pharmacological action of antibiotics –Ideal drug –Influence of factors on performance Drug choice –Cheap –Most Effective –Least toxic